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Double RT-PCR detection kit for H9 subtype avian influenza virus and duck tembusu virus
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A duck Tembusu virus, RT-PCR technology, applied in the field of RT-PCR detection, can solve complex problems
Active Publication Date: 2014-05-14
GUANGXI VETERINARY RES INST
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However, the establishment of a duplex PCR method is much more complicated than that of a single PCR method, which has higher requirements on reagents and primers, and it is necessary to ensure that there is no mutual interference between different primers
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Embodiment 1
[0033] Embodiment 1, design and synthesis of primers
[0034] According to the conserved sequences of the HA gene of H9AIV and the NS2 gene of TMUV in GenBank, DNAStar software was used for multiple sequence alignment, and primer5.0 was used to design primers in the conserved regions. Primers (see Table 1) were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.
[0035] Table 1 Primer information
[0036]
Embodiment 2
[0037] Embodiment 2, establishment of double RT-PCR detection method
[0038] 1. Extraction of nucleic acid and reverse transcription of RNA
[0039]Referring to the instructions of the DNA / RNA extraction kit, extract the DNA of Muscovy duck parvovirus, duck circovirus, duck plague virus and duck embryoallantoic fluid, and simultaneously isolate H5, H9, Muscovy duck reovirus, duck paramyxovirus, The RNA of duck hepatitis virus and TMUV was extracted, and the RNA was reverse-transcribed into cDNA according to the reverse transcription instructions, as follows: 10 μL system was used, and 2 μL of 5× reverse transcription buffer, 10 mmol / L dNTP1 μL were sequentially added to the EP tube , 5U / μL MV 0.5μL, 20U / μL RNase inhibitor 0.5μL, free primer 0.5μL, total RNA template to be tested 2μL, use RNase-free sterilized water to make up the volume to 10μL, put it in the PCR machine after instantaneous separation, 1h at 42°C, 5min at 99°C, and store the prepared cDNA at -30°C for later...
Embodiment 3
[0048] Embodiment 3, the assembly of detection kit
[0049] According to the research results of Examples 1 and 2, a detection kit was assembled for easy use.
[0050] Solution A: PCR reaction solution, containing 25 μL of 2×Taq PCR Mix (purchased from Quanjin. AS111-12), 0.8 μl of H9AIV upstream and downstream primers (both primer concentrations are 50 μmol / mL, and the final concentrations in the reaction system are 0.8 μmol / mL), TMUV upstream and downstream primers each 1.2 μl (primer concentration is 50 μmol / mL, the final concentration in the reaction system is 1.2 μmol / mL), ddH 2 O1.0 μL;
[0051] Solution B: 10 μL each of H9AIV+TMUV template, as a positive control;
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Abstract
The invention discloses a double RT-PCR detection kit for H9 subtype avian influenza virus and duck tembusu virus. The kit comprises two pairs of specific primers. The detection kit disclosed by the invention has the advantages of simplicity in operation, high sensitivity, high specificity, good repeatability and the like; by adopting the detection kit disclosed by the invention, a double PCR detection method for H9 subtype avian influenza virus and duck tembusu virus can be established, which detects and identifies the two pathogens of H9 subtype avian influenza virus and duck tembusu virus at the same time and is applied to the mixed infection of duck tembusu virus resulting from the hypoimmunity caused by the H9 subtype avian influenza virus infection; therefore, the time cost can be saved, and the pollution can be reduced.
Description
technical field [0001] The invention belongs to the technical field of RT-PCR detection, in particular to a double RT-PCR detection kit for H9 subtype avian influenza virus and duck Tembusu virus. Background technique [0002] Duck Tembusu virus (TMUV) was first discovered in 2010 in Fujian, Zhejiang, Jiangsu and other eastern coastal areas of China, and gradually spread to most parts of the country. Infection of ducks with Duck Tembusu virus can lead to a serious drop in egg production of laying ducks, the egg production rate can drop from 90% to less than 10%, or even complete production; infection of ducklings can cause ducklings to stand unsteadily and fall to the ground Can not afford to wait for neurological symptoms, and then lead to the increase of duckling elimination rate, seriously up to 80%, brought great economic losses to the duck industry. Avian influenza is a general term for poultry infections and diseases caused by different subtypes of influenza A viruses...
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